The long-term objectives of our studies are to understand the regulation, function, and disease association of the keratin Intermediate filament proteins, keratin polypeptides 8, 18, 19 and 20 (K8, K18, K19 and K20), that are preferentially expressed in digestive organs. Although the functions of the epithelial-specific keratin proteins remain poorly understood, several skin diseases are caused by mutations in epidermal keratins, and more recently K18 and K8 mutations were identified in patients with cryptogenic cirrhosis. Our overall hypothesis is that studying the posttranslational regulation and dynamics of keratins will provide an effective handle in understanding keratin function and disease pathogenesis. The importance of understanding keratin phosphorylation and glycosylation is based on their general regulatory roles, restricted location to regions of the keratin molecule that provide most of the structural heterogeneity and tissue-specific expression, and hence their likely involvement in dictating tissue-specific functions. Our specific aims and hypotheses are: (i) Study the function of K18 glycosylation in cell culture and transgenic animal models. This is based on the hypothesis that keratin glycosylation, because of its dynamic nature and other features, is likely to play important functional roles. (ii) Study the significance of K18 serine-33 phosphorylation in the pancreas and liver. The hypothesis for this aim is that keratin phosphorylation plays an important role, positively or negatively, in modulating cytoprotective and other possible keratin functions. (iii) Study the significance of Raf kinase and the 14-3-3 protein family association with keratins, and the relationship of Raf/14-3-3, Raf/keratin, and 14-3-3/keratin association. The hypothesis for this aim is that keratins play a role in regulating the availability, localization, or activity of Raf kinase, and possibly other 14-3-3 binding partners. (iv) Study K20 regulation in cell culture and transgenic animal models. The hypothesis for this aim is that the unique cell and differentiation-state distribution of K20 and its relatively high stoichiometry of phosphorylation indicate a differentiation-specific function and a highly regulated keratin species. Our proposed studies are likely to generate important new biologic information regarding keratins in the digestive system, and to help clarify their function and role in human disease.